- Email: [email protected]
Phytic Acid: An Alternative Root Canal Chelating Agent
Basic Research—Technology
Phytic Acid: An Alternative Root Canal Chelating Agent Mohannad Nassar, BDS, MSc,*† Noriko Hiraishi, DDS, PhD,* Yukihiko Tamura, DDS, PhD,‡ Masayuki Otsuki, DDS, PhD,* Kazuhiro Aoki, DDS, PhD,‡ and Junji Tagami, DDS, PhD*† Abstract Introduction: The objectives of this study were to investigate the effect of phytic acid, inositol hexakisphosphate (IP6), as a final rinse on the surface of instrumented root canals and smear-layered flat dentin surfaces treated with sodium hypochlorite (NaOCl) and to evaluate its effect on the viability and alkaline phosphatase activity of osteoblast-like cells (MC3T3E1). Methods: The universally accepted chelating agent EDTA was used as the control in all conducted experiments. Root canals of human canines were instrumented with rotary files and irrigated with 5% NaOCl, followed by a final rinse of 17% EDTA (1 minute), 1% IP6 (1 minute or 30 seconds), or distilled water. NaOCl-treated flat coronal dentin surfaces were also treated with 17% EDTA (1 minute), 1% IP6 (1 minute or 30 seconds), or distilled water. The presence or absence of smear layer was evaluated with scanning electron microscopy. Cell viability and alkaline phosphatase assays were performed to evaluate the effect of IP6 and EDTA on cultured MC3T3-E1 cells. Results: The results demonstrated the ability of IP6 to remove the smear layer from instrumented root canals and flat coronal dentin surfaces. When compared with EDTA, IP6 was less cytotoxic and did not affect the differentiation of MC3T3-E1 cells. Conclusions: IP6 shows the potential to be an effective and biocompatible chelating agent. (J Endod 2015;41:242–247)
I
n endodontics there is always a need for chemomechanical debridement (1). Mechanical debridement results in the formation of smear layer on root canal surfaces. According to the literature, removal of the smear layer before obturation is recommended (2). Sodium hypochlorite (NaOCl) is the main irrigant used during root canal treatment (3); however, NaOCl alone cannot effectively remove the smear layer (4). EDTA has been the most commonly used irrigant for this purpose since 1957 (5) in a concentration of 17% and an application time of 1–5 minutes (6). It is most commonly synthesized on an industrial scale from ethylenediamine, formaldehyde, and sodium cyanide. This method results in the formation of impurities that are detrimental to most applications of this chelating agent (7). This synthetic persistent material is being overused and is considered one of the major organic pollutants discharged in water (8). It is noteworthy that EDTA is used in cosmetic formulations in a concentration less than 2% (9). Because EDTA is not readily biodegradable, there have been some concerns about the leakage of this irrigant into the periapical tissue. Because of these concerns, the extrusion of EDTA beyond the root canal should be avoided (10, 11). Considering these facts, an alternative agent for smear layer removal is warranted, and the search for more biocompatible material to replace EDTA is still going on. Phytic acid (IP6, inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds and bran that contributes in a variety of cellular functions (12). It is also omnipresent in mammalian cells, with a concentration ranging from 10 to 100 mmol/L (13, 14). IP6 can be extracted with low cost from rice bran (15). This agent has multiple negative charges, making it an effective chelator of multivalent cations such as calcium (Ca+2), magnesium, and iron (16, 17). On the basis of these properties, we speculate IP6 to have the potential to replace EDTA as a root canal chelating agent. Thus, the aims of this study were (1) to determine the efficacy of IP6 in removing the smear layer on NaOCl-treated flat coronal dentin surfaces and instrumented root canals dentin and (2) to assess IP6 effect on the viability and alkaline phosphatase (ALP) activity of osteoblast-like cells (MC3T3-E1).
Key Words Chelating agent, EDTA, phytic acid, root canal, smear layer
From the *Cariology and Operative Dentistry, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan; † Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan; and ‡Pharmacology, Department of Bio-Matrix, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan. Address requests for reprints to Dr Noriko Hiraishi, Cariology and Operative Dentistry, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.09.029
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Materials and Methods Smear Layer Removal Effect Flat Coronal Dentin Surface. Human non-carious third molars were used in this part of the study. Flat coronal dentin surfaces of 1-mm thickness were created perpendicular to the tooth’s longitudinal axis by using a slow-speed diamond saw (Isomet Low Speed Saw; Buehler, Lake Bluff, IL) under water lubrication. A smear layer was created on each surface by using 600-grit silicon-carbide paper under water irrigation. The control specimens received only a rinse with distilled water, whereas the other specimens were treated with 5% NaOCl (pH 12) (Wako Pure Chemical Industries, Osaka, Japan) for 5 minutes, followed by 17% EDTA (pH 7.5) (Wako Pure Chemical Industries) for 1 minute, 1% IP6 (pH 1.3) (Wako Pure Chemical Industries) for 1 minute or 30 seconds, or distilled water. All solutions were applied with agitation by using a microbrush. After rinsing with distilled water for 10 seconds, specimens were dehydrated with ascending concentrations of ethanol (25%, 50%, and 75% for 20 minutes, 95% for 30 minutes, and 100% for 60 minutes), followed by immersion in hexamethyldisilazane (Wako Pure Chemical Industries). Specimens were dried overnight inside a covered glass vial and then sputter-coated with gold/palladium and observed under a scanning electron microscope (SEM) (JSM-5310LV scanning microscope; JEOL, Tokyo, Japan) operating at 5 kV.
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Basic Research—Technology
Figure 1. Representative SEM images of effect of different treatments in removing the smear layer from flat coronal dentin surfaces. (A) Smear layer produced by 600-grit silicon-carbide paper. (B) 5% NaOCl applied for 5 minutes. NaOCl was ineffective to completely remove the smear layer. (C) 17% EDTA applied for 1 minute on NaOCl-treated flat coronal dentin surface. EDTA resulted in clean dentinal surface with open dentinal tubules. (D and E) 1% IP6 applied for 1 minute or 30 seconds, respectively, on NaOCl-treated flat coronal dentin surface. IP6 resulted in clean and debris-free surface with widely open dentinal tubules.
Root Canal Surface. Single-rooted maxillary human canines with straight roots and mature apices were used in this part of the study. The experimental setup was followed according to the closed system proposed by Tay et al (18). ProTaper nickel-titanium rotary instruments (Dentsply Maillefer; Ballaigues, Switzerland) were used to prepare and shape the canals according to the manufacturer’s instructions, ending with F3 file. Between each file, the canals were irrigated with 1 mL 5% NaOCl. The canals were rinsed with distilled water before the application of the chelating agents. A final rinse of 1 mL 17% EDTA for 1 minute or 1 mL 1% IP6 for 1 minute or 30 seconds was performed with agitation by using a hand #15 K-file. The canals were then irrigated again with distilled water and dried with absorbent paper points. The control group received no treatment after instrumentation except for a final rinse with distilled water. To facilitate the separation of the root into halves, deep longitudinal grooves were prepared on the external root surface, followed by splitting the root by using a hammer and chisel. The dehydration process for SEM observation was conducted in the same previously mentioned manner. Representative images of JOE — Volume 41, Number 2, February 2015
the middle and apical thirds at 1000 magnification were taken for each group.
Effect on Cell Viability and ALP Activity Cell Viability Assay. The clonal cell line (MC3T3-E1), osteoblast-like cells, established from mouse calvaria, was used in the present study. To each well of 24-well culture plates, MC3T3E1 cells (5 104 cells/well) were placed and incubated for 24 hours in 5% CO2 incubator at 37 C. Six wells were allocated for each test solution. The test solutions included various dilutions (500–10,000 mg/mL culture medium) of either 17% EDTA or 1% IP6. An aliquot of 300 mL of each experimental solution was added to each well and incubated in 5% CO2 incubator at 37 C for 24 hours. Cell culture in fresh medium without experimental solution served as the control. After the incubation time, culture medium was discarded, and cells were washed with 200 mL phosphate buffer solution to prevent any interaction between the test solutions and the colorimetric assay. One hundred microliters of new culture medium Phytic Acid as Chelating Agent
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Figure 2. Representative SEM images of effect of different irrigation regimens on removal of smear layer from middle and apical thirds of instrumented root canals. (A) Smear layer produced on root canal surface after instrumentation and irrigation with 5% NaOCl with final rinse of distilled water. (B and C) Smear layer removal of instrumented root canals that received final rinse of 17% EDTA for 1 minute, middle and apical thirds, respectively. (D and E) Smear layer removal of instrumented root canals that received final rinse of 1% IP6 for 1 minute, middle and apical thirds, respectively. (F and G) Smear layer removal of instrumented root canals that received final rinse of 1% IP6 for 30 seconds, middle and apical thirds; respectively.
was added to each well, and cell viability was measured by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche Diagnostics GmbH, Mannheim, Germany). Twenty microliters of MTT solution was added to each well of the plate and incubated for 3 hours at 37 C. After adding 200 mL dimethyl sulfoxide, the optical density (OD570) was measured with a microplate reader. The morphology of the cultured cells was observed by using phase contrast microscope (1X70; Olympus, Tokyo, Japan).
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ALP Activity Measurement. Cultured MC3T3-E1 cells (5 104 cells/well) were treated with 17% EDTA or 1% IP6 at a concentration of 500 mg/mL culture medium for 1, 7, or 14 days. Both the medium and the test solution were refreshed every 3 days. ALP activity was determined by using ALP Assay Kit (Takara Bio Inc, Shiga, Japan). For statistical analysis, the mean optical density of the MTT test and ALP activity of the control group at 24 hours was set to represent 100%, and results of the experimental groups were expressed as percentages of the control. Statistical analysis was performed by JOE — Volume 41, Number 2, February 2015
Basic Research—Technology
Figure 3. (A) Cytotoxicity of culture medium containing different dilutions of test solutions of 1% IP6 or 17% EDTA on MC3T3-E1 cells after 24 hours of incubation. Cell viability was determined by using MTT assay (n = 6 each group). Two-way ANOVA indicated interaction between irrigants and dilutions. The same lowercase letter indicates no significant difference (P > .05). (B) ALP activity of MC3T3-E1 cells cultured with 500 mg/mL 17% EDTA or 1% IP6 for 1, 7, and 14 days. Data were expressed as percentage of ALP activity in control cells on day 1. Data were analyzed by using one-way ANOVA. The same lowercase letter indicates no significant difference (P > .05).
applying two-way analysis of variance (ANOVA) by using the tested irrigants and dilutions or incubation periods as 2 factors. In case of significance, statistical analyses were performed by Tukey multiple comparison test (alpha = 0.05).
Results Smear Layer Removal Effect Flat Coronal Dentin Surface. Figure 1 shows the topographical changes of smear-layered flat coronal dentin surfaces conditioned with 17% EDTA for 1 minute or 1% IP6 for 1 minute or 30 seconds after an initial treatment with 5% NaOCl for 5 minutes. The 600-grit silicon-carbide paper produced uniform smear layer covering the whole surface (Fig. 1A). The 5-minute application of 5% NaOCl resulted in poor removal of the smear layer (Fig. 1B). The use of 17% EDTA resulted in almost complete removal of the smear layer (Fig. 1C). The complete removal of smear layer was also seen in the case of 1% IP6 applied for 1 minute or 30 seconds (Fig. 1D and E, respectively); however, more widely open dentinal tubules and cleaner surfaces were observed in the case of 1% IP6 in both application times when compared with 17% EDTA. Root Canal Surface. Figure 2 shows the effect of different irrigation regimens on the removal of the smear layer from the middle and apical thirds of instrumented root canals. A smear layer covering the entire surface was shown in canals instrumented and irrigated with 5% NaOCl and a final rinse with distilled water (Fig. 2A). Figure 2B represents the effect of 17% EDTA on the middle third where a clean surface and open dentinal tubules were observed, whereas it showed less effectiveness in the apical third; smear layer was still covering most parts of the surface, with less open dentinal tubules and some debris (Fig. 2C). The use of 1% IP6 for 1 minute or 30 seconds resulted in clean, debris-free, and open dentinal tubules in the middle third (Fig. 2D and F, respectively), whereas the canal wall JOE — Volume 41, Number 2, February 2015
at the apical third was covered with some smear layer and debris (Fig. 2E and G, respectively).
Effect on Cell Viability and ALP Activity The effect of various dilutions of 1% IP6 and 17% EDTA on the viability of MC3T3-E1 cells after 24 hours of exposure is shown in Figure 3A. The data are expressed as percentage of the OD value in the control cells. Two-way ANOVA showed that the factors ‘‘irrigants’’ and ‘‘dilutions’’ were significant (P < .001), and the interaction between these 2 factors was also significant (P < .001). EDTA negatively affected the viability in a dose-dependent manner. The 3 highest dilutions of EDTA caused a significant decrease in cell viability, whereas the lower dilutions were biocompatible. The OD value obtained in 5000 mg/mL EDTA was significantly higher than in 10,000 mg/mL and 7500 mg/mL EDTA (P < .001); however, it was significantly less when compared with the OD values of 2500 mg/mL and 500 mg/mL EDTA (P < .001). The OD values of the latter 2 dilutions were not significantly different (P = .921). All tested dilutions of IP6 had no negative effect on cell viability. The OD value of 500 mg/mL IP6 was significantly higher than those of 10,000 mg/mL (P = .004) and 7500 mg/mL (P = .009) IP6; however, it was not significant when compared with 5000 mg/mL (P = .145) and 2500 mg/mL IP6 (P = .052). For ALP activity assay, two-way ANOVA showed that the ‘‘irrigants’’ and ‘‘incubation times’’ factors were significant (P < .001), but the interaction between these 2 factors was not significant (P = .057). The effect of EDTA and IP6 on ALP activity at 1, 7, and 14 days is shown in Figure 3B. A significant decrease in ALP activity when compared with the control was noticed at 1 day (P = .03) and 14 days (P = .021) in EDTA group. In contrast, IP6 group did not affect the ALP activity when compared with the control group at the 3 incubation times (P > .05). Morphologically, MC3T3-E1 cells in the control media showed the polygonal appearance (Fig. 4A), whereas cells treated with EDTA at the Phytic Acid as Chelating Agent
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Figure 4. Morphologic changes of MC3T3-E1 cells after 24 hours of exposure to test solutions. (A) Control: polygonal-shaped cells. (B–E) Cells treated with 10,000, 7500, 5000, or 2500 mg/mL culture medium of 17% EDTA, respectively. Contracted, spherical morphology and increases in intercellular spaces were observed at 2 highest dilutions (B and C), whereas at 5000 mg/mL, some cells exhibited normal polygonal morphology; however, decreased cellular density and increased intercellular spaces were also observed (D). Normal polygonal morphology of the cells was retained at 2500 mg/mL (E). (F–I) Cells treated with 10,000, 7500, 5000, or 2500 mg/mL culture medium of 1% IP6, respectively. Cells retained normal polygonal morphology at various dilutions of IP6.
2 highest dilutions exhibited contracted, spherical morphology and increases in intercellular spaces, which are indicators of cellular death and decreased proliferation (Fig. 4B and C). For 5000 mg/mL EDTA, some cells maintained the normal morphology; however, cellular density was less, and intercellular spaces were increased (Fig. 4D). The 2500 mg/mL (Fig. 4E) and 500 mg/mL EDTA (data not shown) did not affect the morphology of the cells when compared with the control. For the various dilutions of IP6, cells retained their normal polygonal morphology (Fig. 4F–I).
Discussion Chelating agents are an integral part of root canal therapy. The choice of an irrigation solution should take into consideration its smear layer removal ability and its biocompatibility. In this study, IP6 was able to remove the smear layer from flat coronal dentin surfaces and instrumented root canals and did not show negative effect on the viability and ALP activity of MC3T3-E1 cells. Removal of the smear layer from instrumented root canal surfaces before obturation is a recommended step that is usually done by the use of EDTA solution (2). In this study, EDTA was effective to remove the smear layer from NaOCl-treated flat coronal dentin surfaces and instrumented root canals. This ability resides in the property of ionized 246
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EDTA to chelate Ca+2. The optimum pH for EDTA effectiveness is in the range of 6–10 because higher ratio of ionized to non-ionized molecules can be achieved at higher pH; however, the availability of Ca+2 for chelation decreases at higher pH. When EDTA forms a complex with Ca+2, a proton is released, resulting in decreased pH of the environment in which EDTA loses its efficiency (19). In this study, IP6 was proved to be effective in removing the smear layer from NaOCl-treated flat coronal dentin surfaces and instrumented root canals. IP6 is highly negatively charged molecule that has affinity to Ca+2 (16, 17). Flat coronal dentin surfaces treated with 1% IP6 were cleaner with more widely open dentinal tubules when compared with EDTA. On root canal surfaces, the effect of both IP6 and EDTA in cleaning the apical third was less than that in the middle third, and this is attributed to the anatomy of the former region. The pH of 1% IP6 solution was around 1.2, and this acidity contributed to better Ca+2 extraction. Thus, both the acidity and chelate function of IP6 made it an effective smear layer removal agent. Taking into account that osteoblast cells are necessary element for periapical healing, it is important to assess the effect of irrigants used inside root canals on the viability and ALP activity of these cells because the use of these agents holds the risk of their extrusion beyond the apical foramen. To mimic the clinical situation in which the extruded agent gets diluted once in contact with the periapical tissue, several dilutions JOE — Volume 41, Number 2, February 2015
Basic Research—Technology of 17% EDTA or 1% IP6 were used in this study to test the effect of these irrigants on the viability and ALP activity of MC3T3-E1 cells. The 3 highest dilutions of EDTA caused dramatic decreases in cell viability affecting their morphology, whereas lower dilutions were nontoxic. ALP activity is correlated with the mineralization ability of the osteoblast cells (20); 500 mg/mL 17% EDTA caused significant suppression of ALP activity. The negative effect of EDTA on periapical cells and on the immune reaction has been previously studied. This agent was reported to disrupt the cell membrane structure and the function of macrophages, thus interfering with the healing process (11). The presence of IP6 in the culture medium did not affect the viability, morphology, or ALP activity of the cells in all tested dilutions. IP6 was reported to have a double role in cell culture as an iron chelator and a source of phosphate for cells (21). IP6 protects the cells from oxidative injury through binding to iron, a metal that catalyzes the formation of hydroxyl radicals (22); thus, the results obtained in the present study might be partially attributed to this property. The findings of this study showed the potential of IP6 to be used as a chelating agent in root canal treatment. IP6 proved to be an effective agent in removing the smear layer, while being biocompatible to MC3T3-E1 cells.
Acknowledgments The authors deny any conflicts of interest related to this study.
References 1. Marending M, Paque F, Fischer J, Zehnder M. Impact of irrigant sequence on mechanical properties of human root dentin. J Endod 2007;33:1325–8. 2. H€ulsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment: mode of action and indications for their use. Int Endod J 2003;36:810–30. 3. Zehnder M. Root canal irrigants. J Endod 2006;32:389–98. 4. Torabinejad M, Khademi AA, Babagoli J, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170–5. 5. Nygaard-Ostby B. Chelation in root canal therapy. Odontologisk Tidskrift 1957;65: 3–11.
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6. Calt S, Serper A. Time dependent effects of EDTA on dentine structures. J Endod 2002;28:17–9. 7. Elvers B. Ullmann’s Fine Chemicals. Weinheim: Weinheim, Germany: Wiley-VCH; 2014:647. 8. Sillanp€a€a M. Environmental fate of EDTA and DTPA. Rev Environ Contam Toxicol 1997;152:85–111. 9. Lanigan RS, Yamarik TA. Final report on the safety assessment of EDTA, calcium disodium EDTA, diammonium EDTA, dipotassium EDTA, disodium EDTA, TEAEDTA, tetrasodium EDTA, tripotassium EDTA, trisodium EDTA, HEDTA, and trisodium HEDTA. Int J Toxicol 2002;21(Suppl 2)):95–142. 10. Segura JJ, Calvo JR, Guerrero JM, et al. The disodium salt of EDTA inhibits the binding of vasoactive intestinal peptide to macrophage membranes: endodontic implications. J Endod 1996;22:337–40. 11. Amaral KF, Rogero MM, Fock RA, et al. Cytotoxicity analysis of EDTA and citric acid applied on murine resident macrophages culture. Int Endod J 2007;40:338–43. 12. Raboy V. Myo-inositol-1,2,3,4,5,6-hexakisphosphate. Phytochemistry 2003;64: 1033–43. 13. Szwergold BS, Graham RA, Brown TR. Observation of inositol pentakis- and hexakisphosphates in mammalian tissues by 31P NMR. Biochem Biophys Res Commun 1987;149:874–81. 14. Sasakawa N, Sharif M, Hanley MR. Metabolism and biological-activities of inositol pentakisphosphate and inositol hexakisphosphate. Biochem Pharmacol 1995;50: 137–46. 15. Graf E. Applications of phytic acid. J Am Chem Soc 1983;60:1861–7. 16. Luttrell BM. The biological relevance of the binding of calcium ions by inositol phosphates. J Biol Chem 1993;268:1521–4. 17. Torres J, Dominguez S, Cerda MF, et al. Solution behaviour of myo-inositol hexakisphosphate in the presence of multivalent cations: prediction of a neutral pentamagnesium species under cytosolic/nuclear conditions. J Inorg Biochem 2005;99:828–40. 18. Tay FR, Gu LS, Schoeffel GJ, et al. Effect of vapor lock on root canal debridement by using a side-vented needle for positive-pressure irrigant delivery. J Endod 2010;36: 745–50. 19. O’Connell MS, Morgan LA, Beeler WJ, Baumgartner JC. A comparative study of smear layer removal using different salts of EDTA. J Endod 2000;26:739–43. 20. Beck GR Jr, Sullivan EC, Moran E, Zerler B. Relationship between alkaline phosphatase levels, osteopontin expression, and mineralization in differentiating MC3T3-E1 osteoblasts. J Cell Biochem 1998;68:269–80. 21. Rasmussen L, Toftlund H. Phosphate compounds as iron chelators in animal cell cultures. In Vitro Cell Dev Biol 1986;22:177–9. 22. Xu Q, Kanthasamy AG, Reddy MB. Neuroprotective effect of the natural iron chelator, phytic acid in a cell culture model of Parkinson’s disease. Toxicology 2008;245: 101–8.
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Phytic Acid: An Alternative Root Canal Chelating Agent Mohannad Nassar, BDS, MSc,*† Noriko Hiraishi, DDS, PhD,* Yukihiko Tamura, DDS, PhD,‡ Masayuki Otsuki, DDS, PhD,* Kazuhiro Aoki, DDS, PhD,‡ and Junji Tagami, DDS, PhD*† Abstract Introduction: The objectives of this study were to investigate the effect of phytic acid, inositol hexakisphosphate (IP6), as a final rinse on the surface of instrumented root canals and smear-layered flat dentin surfaces treated with sodium hypochlorite (NaOCl) and to evaluate its effect on the viability and alkaline phosphatase activity of osteoblast-like cells (MC3T3E1). Methods: The universally accepted chelating agent EDTA was used as the control in all conducted experiments. Root canals of human canines were instrumented with rotary files and irrigated with 5% NaOCl, followed by a final rinse of 17% EDTA (1 minute), 1% IP6 (1 minute or 30 seconds), or distilled water. NaOCl-treated flat coronal dentin surfaces were also treated with 17% EDTA (1 minute), 1% IP6 (1 minute or 30 seconds), or distilled water. The presence or absence of smear layer was evaluated with scanning electron microscopy. Cell viability and alkaline phosphatase assays were performed to evaluate the effect of IP6 and EDTA on cultured MC3T3-E1 cells. Results: The results demonstrated the ability of IP6 to remove the smear layer from instrumented root canals and flat coronal dentin surfaces. When compared with EDTA, IP6 was less cytotoxic and did not affect the differentiation of MC3T3-E1 cells. Conclusions: IP6 shows the potential to be an effective and biocompatible chelating agent. (J Endod 2015;41:242–247)
I
n endodontics there is always a need for chemomechanical debridement (1). Mechanical debridement results in the formation of smear layer on root canal surfaces. According to the literature, removal of the smear layer before obturation is recommended (2). Sodium hypochlorite (NaOCl) is the main irrigant used during root canal treatment (3); however, NaOCl alone cannot effectively remove the smear layer (4). EDTA has been the most commonly used irrigant for this purpose since 1957 (5) in a concentration of 17% and an application time of 1–5 minutes (6). It is most commonly synthesized on an industrial scale from ethylenediamine, formaldehyde, and sodium cyanide. This method results in the formation of impurities that are detrimental to most applications of this chelating agent (7). This synthetic persistent material is being overused and is considered one of the major organic pollutants discharged in water (8). It is noteworthy that EDTA is used in cosmetic formulations in a concentration less than 2% (9). Because EDTA is not readily biodegradable, there have been some concerns about the leakage of this irrigant into the periapical tissue. Because of these concerns, the extrusion of EDTA beyond the root canal should be avoided (10, 11). Considering these facts, an alternative agent for smear layer removal is warranted, and the search for more biocompatible material to replace EDTA is still going on. Phytic acid (IP6, inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds and bran that contributes in a variety of cellular functions (12). It is also omnipresent in mammalian cells, with a concentration ranging from 10 to 100 mmol/L (13, 14). IP6 can be extracted with low cost from rice bran (15). This agent has multiple negative charges, making it an effective chelator of multivalent cations such as calcium (Ca+2), magnesium, and iron (16, 17). On the basis of these properties, we speculate IP6 to have the potential to replace EDTA as a root canal chelating agent. Thus, the aims of this study were (1) to determine the efficacy of IP6 in removing the smear layer on NaOCl-treated flat coronal dentin surfaces and instrumented root canals dentin and (2) to assess IP6 effect on the viability and alkaline phosphatase (ALP) activity of osteoblast-like cells (MC3T3-E1).
Key Words Chelating agent, EDTA, phytic acid, root canal, smear layer
From the *Cariology and Operative Dentistry, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan; † Global Center of Excellence (GCOE) Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan; and ‡Pharmacology, Department of Bio-Matrix, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan. Address requests for reprints to Dr Noriko Hiraishi, Cariology and Operative Dentistry, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.09.029
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Materials and Methods Smear Layer Removal Effect Flat Coronal Dentin Surface. Human non-carious third molars were used in this part of the study. Flat coronal dentin surfaces of 1-mm thickness were created perpendicular to the tooth’s longitudinal axis by using a slow-speed diamond saw (Isomet Low Speed Saw; Buehler, Lake Bluff, IL) under water lubrication. A smear layer was created on each surface by using 600-grit silicon-carbide paper under water irrigation. The control specimens received only a rinse with distilled water, whereas the other specimens were treated with 5% NaOCl (pH 12) (Wako Pure Chemical Industries, Osaka, Japan) for 5 minutes, followed by 17% EDTA (pH 7.5) (Wako Pure Chemical Industries) for 1 minute, 1% IP6 (pH 1.3) (Wako Pure Chemical Industries) for 1 minute or 30 seconds, or distilled water. All solutions were applied with agitation by using a microbrush. After rinsing with distilled water for 10 seconds, specimens were dehydrated with ascending concentrations of ethanol (25%, 50%, and 75% for 20 minutes, 95% for 30 minutes, and 100% for 60 minutes), followed by immersion in hexamethyldisilazane (Wako Pure Chemical Industries). Specimens were dried overnight inside a covered glass vial and then sputter-coated with gold/palladium and observed under a scanning electron microscope (SEM) (JSM-5310LV scanning microscope; JEOL, Tokyo, Japan) operating at 5 kV.
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Figure 1. Representative SEM images of effect of different treatments in removing the smear layer from flat coronal dentin surfaces. (A) Smear layer produced by 600-grit silicon-carbide paper. (B) 5% NaOCl applied for 5 minutes. NaOCl was ineffective to completely remove the smear layer. (C) 17% EDTA applied for 1 minute on NaOCl-treated flat coronal dentin surface. EDTA resulted in clean dentinal surface with open dentinal tubules. (D and E) 1% IP6 applied for 1 minute or 30 seconds, respectively, on NaOCl-treated flat coronal dentin surface. IP6 resulted in clean and debris-free surface with widely open dentinal tubules.
Root Canal Surface. Single-rooted maxillary human canines with straight roots and mature apices were used in this part of the study. The experimental setup was followed according to the closed system proposed by Tay et al (18). ProTaper nickel-titanium rotary instruments (Dentsply Maillefer; Ballaigues, Switzerland) were used to prepare and shape the canals according to the manufacturer’s instructions, ending with F3 file. Between each file, the canals were irrigated with 1 mL 5% NaOCl. The canals were rinsed with distilled water before the application of the chelating agents. A final rinse of 1 mL 17% EDTA for 1 minute or 1 mL 1% IP6 for 1 minute or 30 seconds was performed with agitation by using a hand #15 K-file. The canals were then irrigated again with distilled water and dried with absorbent paper points. The control group received no treatment after instrumentation except for a final rinse with distilled water. To facilitate the separation of the root into halves, deep longitudinal grooves were prepared on the external root surface, followed by splitting the root by using a hammer and chisel. The dehydration process for SEM observation was conducted in the same previously mentioned manner. Representative images of JOE — Volume 41, Number 2, February 2015
the middle and apical thirds at 1000 magnification were taken for each group.
Effect on Cell Viability and ALP Activity Cell Viability Assay. The clonal cell line (MC3T3-E1), osteoblast-like cells, established from mouse calvaria, was used in the present study. To each well of 24-well culture plates, MC3T3E1 cells (5 104 cells/well) were placed and incubated for 24 hours in 5% CO2 incubator at 37 C. Six wells were allocated for each test solution. The test solutions included various dilutions (500–10,000 mg/mL culture medium) of either 17% EDTA or 1% IP6. An aliquot of 300 mL of each experimental solution was added to each well and incubated in 5% CO2 incubator at 37 C for 24 hours. Cell culture in fresh medium without experimental solution served as the control. After the incubation time, culture medium was discarded, and cells were washed with 200 mL phosphate buffer solution to prevent any interaction between the test solutions and the colorimetric assay. One hundred microliters of new culture medium Phytic Acid as Chelating Agent
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Figure 2. Representative SEM images of effect of different irrigation regimens on removal of smear layer from middle and apical thirds of instrumented root canals. (A) Smear layer produced on root canal surface after instrumentation and irrigation with 5% NaOCl with final rinse of distilled water. (B and C) Smear layer removal of instrumented root canals that received final rinse of 17% EDTA for 1 minute, middle and apical thirds, respectively. (D and E) Smear layer removal of instrumented root canals that received final rinse of 1% IP6 for 1 minute, middle and apical thirds, respectively. (F and G) Smear layer removal of instrumented root canals that received final rinse of 1% IP6 for 30 seconds, middle and apical thirds; respectively.
was added to each well, and cell viability was measured by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche Diagnostics GmbH, Mannheim, Germany). Twenty microliters of MTT solution was added to each well of the plate and incubated for 3 hours at 37 C. After adding 200 mL dimethyl sulfoxide, the optical density (OD570) was measured with a microplate reader. The morphology of the cultured cells was observed by using phase contrast microscope (1X70; Olympus, Tokyo, Japan).
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ALP Activity Measurement. Cultured MC3T3-E1 cells (5 104 cells/well) were treated with 17% EDTA or 1% IP6 at a concentration of 500 mg/mL culture medium for 1, 7, or 14 days. Both the medium and the test solution were refreshed every 3 days. ALP activity was determined by using ALP Assay Kit (Takara Bio Inc, Shiga, Japan). For statistical analysis, the mean optical density of the MTT test and ALP activity of the control group at 24 hours was set to represent 100%, and results of the experimental groups were expressed as percentages of the control. Statistical analysis was performed by JOE — Volume 41, Number 2, February 2015
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Figure 3. (A) Cytotoxicity of culture medium containing different dilutions of test solutions of 1% IP6 or 17% EDTA on MC3T3-E1 cells after 24 hours of incubation. Cell viability was determined by using MTT assay (n = 6 each group). Two-way ANOVA indicated interaction between irrigants and dilutions. The same lowercase letter indicates no significant difference (P > .05). (B) ALP activity of MC3T3-E1 cells cultured with 500 mg/mL 17% EDTA or 1% IP6 for 1, 7, and 14 days. Data were expressed as percentage of ALP activity in control cells on day 1. Data were analyzed by using one-way ANOVA. The same lowercase letter indicates no significant difference (P > .05).
applying two-way analysis of variance (ANOVA) by using the tested irrigants and dilutions or incubation periods as 2 factors. In case of significance, statistical analyses were performed by Tukey multiple comparison test (alpha = 0.05).
Results Smear Layer Removal Effect Flat Coronal Dentin Surface. Figure 1 shows the topographical changes of smear-layered flat coronal dentin surfaces conditioned with 17% EDTA for 1 minute or 1% IP6 for 1 minute or 30 seconds after an initial treatment with 5% NaOCl for 5 minutes. The 600-grit silicon-carbide paper produced uniform smear layer covering the whole surface (Fig. 1A). The 5-minute application of 5% NaOCl resulted in poor removal of the smear layer (Fig. 1B). The use of 17% EDTA resulted in almost complete removal of the smear layer (Fig. 1C). The complete removal of smear layer was also seen in the case of 1% IP6 applied for 1 minute or 30 seconds (Fig. 1D and E, respectively); however, more widely open dentinal tubules and cleaner surfaces were observed in the case of 1% IP6 in both application times when compared with 17% EDTA. Root Canal Surface. Figure 2 shows the effect of different irrigation regimens on the removal of the smear layer from the middle and apical thirds of instrumented root canals. A smear layer covering the entire surface was shown in canals instrumented and irrigated with 5% NaOCl and a final rinse with distilled water (Fig. 2A). Figure 2B represents the effect of 17% EDTA on the middle third where a clean surface and open dentinal tubules were observed, whereas it showed less effectiveness in the apical third; smear layer was still covering most parts of the surface, with less open dentinal tubules and some debris (Fig. 2C). The use of 1% IP6 for 1 minute or 30 seconds resulted in clean, debris-free, and open dentinal tubules in the middle third (Fig. 2D and F, respectively), whereas the canal wall JOE — Volume 41, Number 2, February 2015
at the apical third was covered with some smear layer and debris (Fig. 2E and G, respectively).
Effect on Cell Viability and ALP Activity The effect of various dilutions of 1% IP6 and 17% EDTA on the viability of MC3T3-E1 cells after 24 hours of exposure is shown in Figure 3A. The data are expressed as percentage of the OD value in the control cells. Two-way ANOVA showed that the factors ‘‘irrigants’’ and ‘‘dilutions’’ were significant (P < .001), and the interaction between these 2 factors was also significant (P < .001). EDTA negatively affected the viability in a dose-dependent manner. The 3 highest dilutions of EDTA caused a significant decrease in cell viability, whereas the lower dilutions were biocompatible. The OD value obtained in 5000 mg/mL EDTA was significantly higher than in 10,000 mg/mL and 7500 mg/mL EDTA (P < .001); however, it was significantly less when compared with the OD values of 2500 mg/mL and 500 mg/mL EDTA (P < .001). The OD values of the latter 2 dilutions were not significantly different (P = .921). All tested dilutions of IP6 had no negative effect on cell viability. The OD value of 500 mg/mL IP6 was significantly higher than those of 10,000 mg/mL (P = .004) and 7500 mg/mL (P = .009) IP6; however, it was not significant when compared with 5000 mg/mL (P = .145) and 2500 mg/mL IP6 (P = .052). For ALP activity assay, two-way ANOVA showed that the ‘‘irrigants’’ and ‘‘incubation times’’ factors were significant (P < .001), but the interaction between these 2 factors was not significant (P = .057). The effect of EDTA and IP6 on ALP activity at 1, 7, and 14 days is shown in Figure 3B. A significant decrease in ALP activity when compared with the control was noticed at 1 day (P = .03) and 14 days (P = .021) in EDTA group. In contrast, IP6 group did not affect the ALP activity when compared with the control group at the 3 incubation times (P > .05). Morphologically, MC3T3-E1 cells in the control media showed the polygonal appearance (Fig. 4A), whereas cells treated with EDTA at the Phytic Acid as Chelating Agent
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Figure 4. Morphologic changes of MC3T3-E1 cells after 24 hours of exposure to test solutions. (A) Control: polygonal-shaped cells. (B–E) Cells treated with 10,000, 7500, 5000, or 2500 mg/mL culture medium of 17% EDTA, respectively. Contracted, spherical morphology and increases in intercellular spaces were observed at 2 highest dilutions (B and C), whereas at 5000 mg/mL, some cells exhibited normal polygonal morphology; however, decreased cellular density and increased intercellular spaces were also observed (D). Normal polygonal morphology of the cells was retained at 2500 mg/mL (E). (F–I) Cells treated with 10,000, 7500, 5000, or 2500 mg/mL culture medium of 1% IP6, respectively. Cells retained normal polygonal morphology at various dilutions of IP6.
2 highest dilutions exhibited contracted, spherical morphology and increases in intercellular spaces, which are indicators of cellular death and decreased proliferation (Fig. 4B and C). For 5000 mg/mL EDTA, some cells maintained the normal morphology; however, cellular density was less, and intercellular spaces were increased (Fig. 4D). The 2500 mg/mL (Fig. 4E) and 500 mg/mL EDTA (data not shown) did not affect the morphology of the cells when compared with the control. For the various dilutions of IP6, cells retained their normal polygonal morphology (Fig. 4F–I).
Discussion Chelating agents are an integral part of root canal therapy. The choice of an irrigation solution should take into consideration its smear layer removal ability and its biocompatibility. In this study, IP6 was able to remove the smear layer from flat coronal dentin surfaces and instrumented root canals and did not show negative effect on the viability and ALP activity of MC3T3-E1 cells. Removal of the smear layer from instrumented root canal surfaces before obturation is a recommended step that is usually done by the use of EDTA solution (2). In this study, EDTA was effective to remove the smear layer from NaOCl-treated flat coronal dentin surfaces and instrumented root canals. This ability resides in the property of ionized 246
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EDTA to chelate Ca+2. The optimum pH for EDTA effectiveness is in the range of 6–10 because higher ratio of ionized to non-ionized molecules can be achieved at higher pH; however, the availability of Ca+2 for chelation decreases at higher pH. When EDTA forms a complex with Ca+2, a proton is released, resulting in decreased pH of the environment in which EDTA loses its efficiency (19). In this study, IP6 was proved to be effective in removing the smear layer from NaOCl-treated flat coronal dentin surfaces and instrumented root canals. IP6 is highly negatively charged molecule that has affinity to Ca+2 (16, 17). Flat coronal dentin surfaces treated with 1% IP6 were cleaner with more widely open dentinal tubules when compared with EDTA. On root canal surfaces, the effect of both IP6 and EDTA in cleaning the apical third was less than that in the middle third, and this is attributed to the anatomy of the former region. The pH of 1% IP6 solution was around 1.2, and this acidity contributed to better Ca+2 extraction. Thus, both the acidity and chelate function of IP6 made it an effective smear layer removal agent. Taking into account that osteoblast cells are necessary element for periapical healing, it is important to assess the effect of irrigants used inside root canals on the viability and ALP activity of these cells because the use of these agents holds the risk of their extrusion beyond the apical foramen. To mimic the clinical situation in which the extruded agent gets diluted once in contact with the periapical tissue, several dilutions JOE — Volume 41, Number 2, February 2015
Basic Research—Technology of 17% EDTA or 1% IP6 were used in this study to test the effect of these irrigants on the viability and ALP activity of MC3T3-E1 cells. The 3 highest dilutions of EDTA caused dramatic decreases in cell viability affecting their morphology, whereas lower dilutions were nontoxic. ALP activity is correlated with the mineralization ability of the osteoblast cells (20); 500 mg/mL 17% EDTA caused significant suppression of ALP activity. The negative effect of EDTA on periapical cells and on the immune reaction has been previously studied. This agent was reported to disrupt the cell membrane structure and the function of macrophages, thus interfering with the healing process (11). The presence of IP6 in the culture medium did not affect the viability, morphology, or ALP activity of the cells in all tested dilutions. IP6 was reported to have a double role in cell culture as an iron chelator and a source of phosphate for cells (21). IP6 protects the cells from oxidative injury through binding to iron, a metal that catalyzes the formation of hydroxyl radicals (22); thus, the results obtained in the present study might be partially attributed to this property. The findings of this study showed the potential of IP6 to be used as a chelating agent in root canal treatment. IP6 proved to be an effective agent in removing the smear layer, while being biocompatible to MC3T3-E1 cells.
Acknowledgments The authors deny any conflicts of interest related to this study.
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